A fundamental requirement of cell lines is accurate characterization and exclusion of cross-contamination, whether by microorganisms or other cells. Cross-contaminated or misidentified cultures might yield invalid data and waste scarce resources (1).
First reports on cross-contaminated cultures appeared soon after the worldwide spreading and utilization of cell lines, as exemplified by the “HeLa-story” (2, 3). Although this problem may be chronic, and the scientific community adequately warned about this “hidden danger” (4-6), the need for authentication seems remains insufficiently appreciated rendering quality controls an essential part of current good laboratory practice (1, 7-9).

Species Identification of Animal Cell Lines

Thanks to negative publicity initially prompted by a series of DSMZ publications highlighting the phenomenon, cell line cross contamination (CLCC) is now accepted as a major problem in cell culture. CLCC usually involves the seeding - presumably via shared pipettes, reagents etc - and eventual replacement of fledgling “new” cell line cultures by faster growing established cell lines. While short tandem repeat (STR)-DNA profiling detects cross-contamination among human cell lines, detection among animal cell lines at the DSMZ  is now performed by DNA-barcoding, a method involving PCR-amplification and sequencing of mitochondrial  DNA, followed by comparing the sequence in international data bases.

The Past

At the DSMZ, isoenzyme analysis, partially supplemented by cytogenetics, was used for identifying species in mammalian cell lines until 2000 (10). Since then, we are using DNA-bases methods. While DNA-fingerprinting techniques are used for the detection of individual human samples (11, 12) and as an international standard for authentication of human cell lines (13-15), animal cell lines derive from genetically homogeneous lab-bred individuals and require a different approach. One early study described PCR-assay using a single (alu) primer for distinguishing mouse and Chinese hamster cell lines (16). Based on this work, the DSMZ and other cell repositories have developed a PCR-based method for verifying species in commonly used cell lines (17-20). Our first approach was to introduce informative primer sets targeting exon or intron sequences of the β-globin and β-actin gene verifying the presumptive origin species of human, mouse, rat and hamster (17).

Also, we developed a PCR, with only one primer pair of repetitive sequences for hamster, enabling us to distinguish in one assay the above mentioned rodents plus human cells (21; Figure 1). By using fluorescence-labelled primers in connection with capillary electrophoresis, the length of the PCR-amplicons can be determined more precisely. Also, since the detection limit of capillary electrophoresis compared to standard agarose gel electrophoresis is much lower, even minute amounts of the respective DNAs can be detected (Figure 2).

In life sciences, the most ubiquitous species of origin are rodent and human (Figure 3). However, for cell lines used in other disciplines, for example in veterinary medicine or in biotechnology, it is equally important to verify domestic animal species and to exclude cross contamination with other (animal) cell lines.


Since the mitochondrial genomes of a large number of animals are sequenced, it was obvious to develop PCR assays targeting mitochondrial DNA to evaluate samples of a larger number from different species. Like colleagues from Japanese and US cell banks (22, 23), we applied standard and multiplex-PCR for the analyses of the number of almost twenty animal cell lines (24; Figure 4 as an example of some livestock animals).

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The Present: DNA Barcoding

The development of fast and robust sequencing of even tiny amounts of DNA initiated a new area of taxonomy combining PCR amplification and sequencing. By using degenerative primers a  ca. 650 base-pairs region (the ”barcode”) of the mitochondrial Cytochrome C Oxidase I (CO1) from numerous taxa, including “all” species of fish, amphibians, reptiles,  birds, mammals and even insects can be amplified and subsequently sequenced.  The data are then compared with those of a public sequence database to assign the unknown sample to a known species. By now, all deposited animal cell lines have been re-evaluated by the barcoding method and almost all of the originally supposed species have been confirmed. In case of the very low number of the unmatched species, the database suggested a (very) close related species or even a subspecies.

Selected References

  1. Stacey GN, Masters JR, Hay RJ, Drexler HG, MacLeod RAF and Freshney RI: Cell contamination leads to inaccurate data: We must take action now. Nature 403: 356 (2000).
  2. Gartler SM: Apparent HeLA contamination of human heterodiploid cell lines. Nature 217: 750-751 (1968).
  3. Nelson-Ress WA , Flandermeyer RR: HeLa cultures defined. Science 191: 96-98 (1976).
  4. Stevenson R: Development of cell banking in the US 1960-1985: a strategic approach to quality control. Adv Cell Cult 51: 267-288 (1987).
  5. Nelson-Rees WA, Daniels DW and Flandermeyer RR: Cell cross-contamination in cell cultures. Science 212: 446-452 (1981).
  6. Drexler HG, Dirks WG, MacLeod RAF: False human hematopoietic cell lines: Cross-contaminations and misinterpretations. Leukemia 13: 1601-1607 (1999).
  7. Zoon KC: Points to consider in the characterization of cell lines used to produce biologicals. Rockville, MD: Center for Biological Evaluation and Research. Food and Drug Administration: 7-8 (1993).
  8. MacLeod RAF, Dirks WG and Drexler HG: Persistent use of misidentified cell lines and its prevention. Genes Chromosomes Cancer 33: 103-105 (2002).
  9. Masters JR: False cell lines: The problem and the solution. Cytotechnology 39: 17-22 (2002).
  10. Steube KG, Grunicke D, Drexler HG: Isoenzyme analysis as a rapid method for the examination of the species identity of cell cultures. In Vitro Cell Dev Biol 31A: 115-119 (1995).
  11. Jeffreys AJ, Wilson V and Thein SL: Individual-specific fingerprints of human DNA. Nature 316: 76-79 (1985).
  12. Stacey GN, Bolton BJ and Doyle A: DNA fingerprinting transforms the art of cell authentication. Nature 357: 261-262 (1992).
  13. Dirks WG, MacLeod RAF, Jaeger K, Milch H and Drexler HG: First searchable database for DNA profiles of human cell lines: sequential use of fingerprint techniques for authentication. Cell Mol Biol 45: 841-853 (1999).
  14. Masters JR, Thomson JA, Daly-Burns B, Reid YA, Dirks WG, Packer P, Toji LH, Ohno T, Tanabe H, Arlett CF, Kelland LR, Harrison M, Virmani A, Ward TH, Ayres KL and Debenham PG: Short tandem repeat profiling provides an international reference standard for human cell lines. Proc Natl Acad Sci USA 98: 8012-8017 (2001).
  15. Dirks WG, MacLeod RAF, Nakamura Y, Kohara A, Reid Y, Milch H, Drexler HG, Mizusawa H: Cell line cross-contamination initiative: An interactive reference database of STR profiles covering common cancer cell lines. Int J Cancer 126: 302-304 (2010)
  16. Thacker J: Fingerprinting of mammalian cell lines with a single PCR primer. BioTechniques 16: 252-253 (1993).
  17. Steube KG, Meyer C, Uphoff CC, Drexler HG: A simple method using beta-globin polymerase chainreaction for the species identification of animal cell lines- a progress report. In Vitro Cell Dev Biol 39A: 468-475 (2003).
  18. Parodi P, Aresu O, Bini D, Lorenzini R, Schena F, Visconti P, Cesaro M, Ferrera D, Andreotti V, Ruzzon T: Species identification and confirmation of human and animal cell lines: a PCR based method. BioTechniques 32: 432-440 (2002).
  19. Liu MY, Lin SC, Liu H, Candal F and Vafai A: Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing. In Vitro Cell Dev Biol 39A: 424-427 (2003).
  20. Stacey GN, Hoelzl H, Stephenson JR and Doyle A: Authentication of animal cell cultures by direct visualization of repetitive DNA, aldolase gene PCR and isoenzyme analysis. Biologicals 25: 75-85 (1997).
  21. Steube KG, Koelz AL, Drexler HG: Identification and verification of rodent cell lines by polymerase chain reaction. Cytotechnology 56: 49-56 (2008).
  22. Ono K, Motonobu S, Yoshida T, Ozawa Y, Kohara A, Takeuchi M, Mizusawa H, Sawada H: Species identification of animal cells by nested PCR targeted to mitochondrial DNA. In Vitro Cell Dev Biol 43A: 168-175 (2007).
  23. Cooper JK, Sykes G, King S, Cottrill K, Ivanova NV, Hanner R, Ikonomi P: Species identification in cell culture: a two-pronged molecular approach. In Vitro Cell Dev Biol 43A: 344-351 (2007).
  24. Steube KS, Koelz AL, Uphoff CC, Drexler HG, Kluess J Steinberg P: The necessity of identity assessment of animal intestinal cell lines: A case report. Cytotechnology 64: 373-378 (2012).